WO2008089519A1

WO2008089519A1 - Human papillomavirus screening method

Info

Publication number: WO2008089519A1
Application number: PCT/AU2008/000087
Authority: WO
Inventors: Philip Felix Tynan; Victor Agustin Franco
Original assignee: Symbion Pathology Pty Limited
Priority date: 2007-01-25
Filing date: 2008-01-25
Publication date: 2008-07-31

Abstract

The present invention relates to methods for screening for human papilloma viruses (HPV), in particular for screening for human genital papilloma viruses which are associated with neoplasia such as cervical cancer. The methods comprise the amplification and detection of HPV in biological samples using PCR with HPV type-specific primers. The methods may comprise an additional step of screening for the presence of HPV which may be prior to the amplification and detection step with HPV type-specific primers. The methods of the invention allow the detection o multiple HPV types in a single biological sample. HPV type-specific primers and probes are disclosed as well as consensus primers. Kits, components and compositions for use in the methods of the invention are also disclosed.

Description

Human papillomavirus screening method Technical Field

The present invention relates to methods for screening for human papilloma viruses, in particular methods for screening for human genital papilloma viruses which are associated with neoplasia, such as cervical cancer. Kits, components and compositions for use in the invention are also provided.

Background of the Invention

Papillomaviruses are a diverse group of small, closed circle non-enveloped, double stranded DNA viruses that infect a wide range of animals. They were first identified in the early 20th century, when it was discovered that skin warts, or papillomas, could be transmitted between individuals. In 1935, Rous demonstrated that the papillomavirus could cause also skin cancer in infected rabbits.

Human genital papillomaviruses are amongst the most prevalent sexually transmitted human pathogens. Most genital HPV infections in women produce transient squamous cell abnormalities of the cervix that resolve completely, and so the probability of any one HPV infection progressing to cervical cancer is quite small. Nevertheless, Human Papillomavirus (HPV) is associated with 99.7% of squamous cell cancers of the cervix. Worldwide, HPV-associated cervical carcinoma is the second most common form of cancer in women, with nearly 471,000 new cases and 233,000 deaths annually. HPVs are also associated with anal neoplasia, especially in subjects with concurrent HIV infections.

Papillomaviruses are classified by nucleic acid sequence homology rather than serologic reactivity. More than one hundred types of HPV have been identified. The approximately 50 anogenital HPV types have been segregated into low, medium, and high risk types according to their oncogenic potential. Types 6 and 11 are the most common low risk types; however, they are associated with benign diseases, the most common being genital warts. Types 16 and 18 have been reported as the most common high risk types, being the most frequent types found in cervical cancers. There are around 20 types that are considered "high risk". The current screening method of choice for HPV-associated cervical cancer or cancer precursors is by Papanicolaou (Pap)-cytology. The Pap test identifies abnormal cervical cells from HPV infection lesions which may persist for 12-18 months following infection. Cervical cancer develops from these precursor lesions which, depending on the extent of disruption of epithelial differentiation, may be graded I-III according to the cervical intraepithelial neoplasia (CIN) scale. CIN III represents the immediate precursor to cervical cancer. The relatively low sensitivity of the Pap test and the absence demonstration by the pap test of the causative agent have substantially hindered a further reduction of this form of cancer, although the recent development of an effective vaccine to two common types of HPV offers promise in reducing the incidence of HPV-associated neoplasia.

An alternative to screening for cervical cells of altered phenotype is to test directly for HPV types which are associated with the majority of cervical neoplasms. Direct testing for HPV relies extensively on molecular biology techniques using nucleic acid probes, because HPVs cannot be cultured in vitro and currently there is no suitable serological test for HPV infection.

Several molecular methodologies have been applied to the detection of HPV, including Polymerase Chain Reaction (PCR) and restriction enzyme analysis, DNA sequencing, branch-chain DNA analysis (bDNA) and reverse blotting. The Hybrid Capture™2 (HC2, Digene Co., Gaithersburg, MD, USA) technique has been employed to detect the most clinically relevant high-risk oncogenic HPV types, namely types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Other test kits which utilise the HC2 technique are offered to differentiate between low-risk types such as 6, 11, 42, 43, or 44 and high-risk types.

The HC2 technique utilises cocktails of specific RNA probes which are directed toward target HPV DNA sequences. IfHPV DNA sequences are present, they will hybridise with the specific RNA probes to form a DNA/RNA hybrid, which is captured and detected with an antibody to DNA/RNA hybrids, and labelled with a second DNA/RNA hybrid antibody for signal amplification in a microplate chemiluminescence detection system. This technique does not distinguish which of the HPV types are detected from amongst the cocktail of probes are present in a positive sample. In addition, false negative results may occur if the virus number is low.

An alternative to hybrid capture is an in situ hybridization test using a cocktail of fluorescein-tagged DNA probes. This technique may be used on histology or liquid based pap test slides with separate probe kits for oncogenic and non-oncogenic HPV types. Stained target cells, signifying the presence of HPV infection, are identified by observation under a microscope. Polymerase Chain Reaction (PCR) based systems which use consensus or degenerate primer sequences to allow the amplification and identification of many of the high oncogenic-risk HPV types offer high sensitivity, but do not reliably allow the identification of multiple HPV types in a single sample. In addition, such primers often substantially underestimate the prevalence of some HPV types.

Another system utilises PCR of HPV DNA on a DNA microarray, which effectively functions as a fluorescent PCR-ELISA assay, where the microplate wells are analysed by a laser plate scanner. This system detects 24 types of HPV by the PCR amplification of a 350 bp fragment from the HPV El gene using 20 forward primers. Only one reverse primer is used, as the assay exploits a region of the HPV genome where the reverse primer is highly conserved across HPV types. One weakness of this approach is that reagent limitations in the microplate wells do not reliably allow multiple co-infections to be detected, and at best possibly two co-existing HPV types might be detected in a single sample. Summary of the Invention

In one aspect there is provided a method of identifying and distinguishing between human papilloma virus types in a biological sample, comprising the amplification and detection of human papilloma virus DNA in the biological sample using PCR with human papilloma virus type-specific primers. In one embodiment, this method comprises the additional step of screening for the presence of human papilloma virus in the biological sample. The screening step may take place before the step of amplification and detection of human papilloma virus DNA in the biological sample using PCR with human papilloma virus type-specific primers.

In a further embodiment the method is capable of identifying and distinguishing between multiple human papilloma virus types in a sample infected with more than one human papilloma virus type, and may be capable of identifying and distinguishing between multiple high oncogenic risk human papilloma virus types in a sample infected with more than one human papilloma virus type. In a particular embodiment, the human papilloma virus type-specific primers include any one or more nucleotide sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

In particular embodiment, the amplification of human papilloma virus DNA is identified by one or more probe nucleotide sequences selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and

SEQ ID NO:42. The one or more probe nucleotide sequences may comprise a minor groove-binding moiety.

The biological sample may be a cervical sample or an anogenital sample. Where the biological sample is a cervical sample, it may be collected using a tampon.

In one embodiment, the screening for the presence of human papilloma virus in the biological sample is by amplification and detection of human papilloma virus DNA. In a particular embodiment the amplification and detection of human papilloma virus DNA is by real time PCR. The amplification of human papilloma virus DNA may utilise at least one consensus primer for multiple human papilloma virus types, such as primers selected from the nucleotide sequence SEQ ID NO: 1 and/or SEQ ID NO:2.

Also provided is a kit for identifying and distinguishing between human papilloma virus types in a biological sample by PCR, comprising human papilloma virus type- specific primers. In one embodiment, the human papilloma virus type-specific primers are selected from the group consisting of any one or more of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

Also provided is a method of screening for the presence of human papilloma virus in a biological sample by amplification and detection of human papilloma virus DNA using PCR and at least one human papilloma virus consensus primer which is selected from the nucleotide sequence SEQ ID NO:1 and/or SEQ ID NO:2. In a particular embodiment, this method comprises the step of identifying and distinguishing between human papilloma virus types in the biological sample by the amplification of human papilloma virus DNA using PCR with human papilloma virus type-specific primers.

Also provided is a method of diagnosis of HPV infection in a subject, comprising obtaining a biological specimen which is suspected of containing HPV from a subject and amplifying and detecting human papilloma virus DNA using PCR with human papilloma virus type-specific primers. In one embodiment the diagnosis of HPV infection identifies a predisposition for HPV-associated neoplasia.

Also provided is a method of identifying a persistent or recurring HPV infection, comprising comparing the HPV types present in two or more temporally separated biological samples from a subject, wherein the HPV types are identified and distinguished by amplifying and detecting type-specific human papilloma virus DNA in the biological samples using PCR with human papilloma virus type-specific primers.

Also provided is a method of screening for HPV types in a biological sample, comprising

(i) screening the biological sample for the presence of HPV using real time PCR and consensus primers specific to HPV; and

(ii) where HPV was detected in (i) screening for HPV types using PCR and HPV type-specific primers.

Also provided is a consensus primer for screening for the presence of HPV in a biological sample, the consensus primer consisting of a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2.

Also provided is an HPV type-specific primer for the identification of specific HPV types in a biological sample, the HPV type-specific primer consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

Definitions

In the context of this specification, the term polymerase chain reaction or PCR is intended to broadly encompass the varied techniques which utilise a primer or primers to selectively direct the action of a DNA polymerase on a template sequence to repeatedly produce (or amplify) specific DNA sequences. PCR commonly utilises pairs of primers which bind both strands of the DNA duplex, as this allows for the exponential amplification of the selected DNA strands; however it will be understood that asymmetric or single strand PCR may also be carried out using primers to only one DNA strand, although such techniques only allow for the linear amplification of the desired DNA sequence. Reverse transcription PCR (RT-PCR) may also be used in the context of the present invention to identify HPV RNA sequences, convert them to cDNA and then allow for PCR amplification. Real time PCR, in which the process of amplification may be monitored and quantified using DNA binding dyes or labelled specific nucleotide sequence probes is also contemplated.

In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising" such as "comprise" and "comprises" have correspondingly varied meanings. Throughout this specification, reference to "a" or "one" element does not exclude the plural, unless context determines otherwise. For instance, reference to "a HPV" should not be read as excluding the possibility of multiple HPV.

Detailed Description of the Preferred Embodiments

The present inventors have developed a method which allows the sensitive and specific identification and distinguishing of different human papilloma virus types in a biological sample, and in particular human papilloma virus types associated with neoplasia.

The methods described herein allow for the identification and distinguishing between different types of HPV present together in a biological sample, by using primers which are specific for each of the HPV types of interest to selectively amplify specific HPV DNA by PCR. The methods described herein thus are suitable for not only identifying the presence of HPV in the biological sample, but also identifying which HPV types are present.

The ability to distinguish between the presence of individual HPV types in a biological sample from a subject, for example a cervical sample, is advantageous for several reasons. The presence of an HPV infection of the cervix by a high oncogenic risk HPV type which does not resolve within the normal time course of HPV infection (on average an HPV infection of the cervix takes two years to resolve, in the absence of re- infection) is suggestive that a subject may be at risk of developing an HPV-associated neoplasia. Screening methods which do not distinguish between multiple HPV types in concurrent HPV infections are unable to identify whether the presence of one or more HPV types in a cervical sample over an extended time represents an extended infection with those HVP types, or a number of transient, resolving infections by several HPV types.

In addition, it may be advantageous to be able to identify the presence of a small number of specific HPV types in a subject, for example to track the response of a subject or a population of subjects to immunization against certain HPV strains. The use of HPV type-specific primers allows for specificity in detecting different

HPV types, and may avoid or ameliorate some of the disadvantages of other HPV techniques. In particular, the use of HPV type-specific primers allows the amplification of different HPV type DNA in different reaction vessels, and consequently minimises the risk of any one amplification and detection reaction dominating others and masking the presence of multiple HPV types. Depending on the number of specific primers which are used, the methods described herein provide the ability to identify 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 different HPV types and distinguish between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 different HPV types present in a single biological sample.

It will be recognised that the product of the PCR reaction in amplifying a specific

DNA sequence may be detected in numerous ways, any one of which is contemplated in the present invention. The use of DNA-binding reporting dyes, such as ethidium bromide or SYBR green which bind to double stranded DNA are a convenient method by which the presence of amplified DNA product may be detected. The use of specific probes, such as nucleotide sequences which are complementary for the amplified product also allows the identification of amplification products. Such probes may be labelled, for example with a fluorescent reporter dye with a fluorescence quencher moiety which quenches the reporter dye fluorescence until the probe hybridises with the PCR amplification product, allowing for a real time assessment of amplification. Real time PCR also offers the advantage of being readily quantitative.

The use of variously labelled different probes in PCR, including real time PCR, allows for multiplexing of amplification reactions, so that two or more amplifications may be carried out, if desired, in a single reaction vessel. The human papilloma virus types contemplated by the present invention are the types associated with anogenital infection, and in particular any one or more types which are associated with high oncogenic risk, as evidenced by their association with neoplasia such as cervical cancer and anal or anogenital neoplasia. High oncogenic risk HPV types are considered to be types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 70, 73 and 82.

The biological sample may be any sample which contains or potentially contains HPV DNA in a form suitable for amplification and detection which is taken from a subject. For the examination of HPV infections of the cervix, for example, techniques used to collect cervical cell samples for other screening tests, such as pap-tests or HC2 techniques, using any appropriate collection procedure or device, including swabs, spatula scrapings, or specialised brushes to sample cells of the cervical epithelium may be suitable for producing the biological sample. Through the use of methods of the invention, relatively small numbers of infected cells or HPV particles are suitable to identify the presence of HPV. Thus samples of urine or liquid washes of the cervical surface may be used to collect the biological sample. Similarly, urethral swabs or other anogenital swabs or washes from either male or female subjects could be used to provide the biological sample. In females, paraffin-embedded tissue or biopsy tissue from the cervix or "thin- preps" may also be used.

The present inventors have also identified that suitable samples of cervical cells for use in the present methods may be obtained from tampons. The use of tampons avoids much of the discomfort which may be associated with other cervical cell sampling procedures, and provides the opportunity for the subject of the screening test to submit samples to a collection and testing facility without the assistance of a medical professional, as such samples may be forwarded to a collection or testing facility in the mail. The inventors have identified that it is possible to successfully test such samples even after delays of several days after collection, or alternatively, the tampons may be treated in a manner suitable for preservation of the sample, such as by immersion in a preservative solution to prevent DNA breakdown.

In particular embodiments, the methods of the present invention utilise the amplification of HPV DNA from a biological sample. Methods for identifying and distinguishing between HPV types in a sample utilise one or more primers, each of which are specific to a single HPV type, and each of which therefore promotes the amplification of only a sequence of DNA which is characteristic of that HPV type. The functional requirements for primers for the amplification of DNA by PCR are well known in the art (see for example Prezioso V, General Notes on Primer design in PCR <http://www.eppendorfna.com/applications/PCR _appl_primer.asp>, the entire contents of which is incorporated herein by cross reference). In addition to the primer sets having sequences described herein, other HPV-type specific primer sets which may be used include the GP-PCR sequences of Szuhai et al. (2001) Am J Pathol 159(5): 1651-1660, the E6/E7 sequences of Flores-Munguia et al. (2004) J Molec Diag 6(2):115-124, the simultaneous quantification sets of Moberg et al. 2003 J Clin Microbiol 41(7): 3221-3228 and the nested amplification sets targeting El and Ll of Ylitalo et al. (1995) J Clin Microbiol 33(7): 1822-1828.

Although single primers rather primer pairs could be used for the amplification reactions in the present methods, the use of primer pairs is preferred.

In order to determine whether any given primer or primer pair is specific for a single HPV type the primer or primer pair may be tested against publicly available nucleotide sequence databases, for example as accessed through the National Center for Biotechnology Information (NCBI), National Library of Medicine, Bldg 38 A, Bethesda, MD20894, USA, to determine probe specificity.

In a preferred embodiment, the present inventors have utilised pairs of primers, each of which are specific for particular high oncogenic risk HPV types. The sequences of these primers are described in SEQ ID NOS: 3 to 36 in Example 3. It will be understood, however, that the substitution of these specific primers with other primers specific for particular HPV types would not materially alter the working of the invention. Furthermore, additional primers specific to other HPV types not described herein may readily be used to broaden the range of HPV types which may be identified by the methods of the invention.

The "consensus primers" described herein refer to primers which are specific for HPV DNA, but which are able to promote the amplification of DNA of many HPV types. Briefly, consensus primers for HPV were designed manually, by comparison of primer base homologies based on the paper by Kleter et al. (American Journal of Pathology 1998; 153: 1731-1739). The consensus primers recognise the majority of relevant HPV types due to flexibility/degeneracy in the DNA sequence. Other consensus primers may be used, provided that their ability to recognise relevant HPV strains is confirmed using samples with known HPV type infections. The present inventors have also utilised a pair of consensus primers which amplify HPV types 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 71, and 74 (as well as types 70, 73 and 82 which were not included in the study by Kleter et al.) for screening for the presence of HPV in a sample. The sequences of these primers are described in Example 2 herein in SEQ ID NOS: 1 and 2. It will be understood, however, that the substitution of these consensus primers with other consensus primers, or the use of more than one pair of consensus primers, or one or more unpaired consensus primers would not materially alter the working of the invention.

In the optional screening step of this method, the presence of HPV is identified in the biological sample, although the screening step does not necessarily identify which of the HPV type or types are present. Thus the result of the screening step will be recognition of either the presence or the absence of HPV. In one embodiment, the screening step identifies the majority of anogenital HPV types. In another embodiment, the screening step identifies the majority of high oncogenic risk HPV types, and may also identify low risk HPV types. Thus the screening step allows for the selection of biological samples in which it is worthwhile to further characterise the HPV type or types present in the sample. The screening step may also be used to confirm a positive result from the step of identification and distinguishing the HPV types. Thus in one embodiment the screening step takes place before the identifying and distinguishing between HPV types in a sample. It will be recognised that there are numerous methods available in the art to screen for the presence of HPV in a sample, and it is contemplated that many such methods, such as the HC2 technique, DNA microarrays, High-Resolution Melting Analysis or PCR may be used for the screening step.

Real-time PCR methodologies are particularly advantageous where large numbers of samples are to be screened because they lend themselves to automation. Accordingly, in a preferred embodiment, the sample is screened by real time PCR using consensus primers for HPV, which advantageously allows the rapid and sensitive amplification and detection of the presence of the majority of HPV sequences. Alternatively, by selection of appropriate consensus primer sequences it may be possible to selectively screen for high oncogenic risk HPV rather than low risk HPV, or vice versa.

Suitable consensus primers for real time PCR screening of HPV are described herein as SEQ ID NOS: 1 and 2. These consensus sequences identify the majority of high oncogenic risk and low risk HPV types. The present inventors have identified that real time PCR using the DNA-binding green dye SYTO^® 9 (Molecular Probes, Eugene, OR, USA, Cat # S-34854; as a reporter of dsDNA was particularly advantageous, as it binds DNA at higher concentrations than SYBR Green, is usable in High-resolution Melting Analysis, and is significantly less toxic, and accordingly in a preferred embodiment the use of this dye is contemplated for PCR reactions for identifying and distinguishing between HPV types, and/or for PCR reactions for screening for the presence of HPV.

Examples

The following examples are intended to serve to illustrate this invention and should not be construed as limiting the general nature of the disclosure of the description throughout this specification.

Example 1. Cervical or anogenital specimen sampling

The present method is applicable to samples obtained by different techniques. Where an anogenital sample is to be taken, a simple swab may be used to collect a representative sample of epithelial cells. Ideally dry swabs are used, but alternatively Charcoal, Stuart's and Viral swabs may be used. i) Elution of HPV particles from Tampons

Where a tampon is used to provide a cervical sample, either synthetic (such as nylon or rayon) or natural fibre (such as cotton) material may be used. Samples may be obtained at any stage of the menstrual cycle except during the actual onset of menses themselves, as the specimen must be substantially blood-free. The tampon may be briefly inserted and removed, or it may be inserted and left in situ for several hours, although ideally less than 12 hours. The insertion and removal of the tampon should be with clean hands, and the used tampon should be placed in a new plastic bag for transport to the collection or testing facility.

For sample preparation, all tampons were manipulated with sterile tweezers which were flamed between samples to avoid cross contamination. An approximately 1 cm³ piece was cut from a region from each tampon which was suspected of bearing cervical cells. The selected piece of tampon was completely immersed in 1ml of DNA- free deionized, distilled water in a sterile sample tube and vigorously vortexed for 5 seconds. The tampon piece was squeezed inside the sample tube to release cervical cells and HPV particles into the deionized water. The sample tubes were sealed and the outsides of the tubes decontaminated with RNase A WAY (Molecular Bio products, Inc). H) Elution of HPV particles from Swabs

The swab from a subject was immersed into a sample tube containing 1 ml of DNA- free distilled, deionised water, and the swab was gently agitated for 10 seconds to release cervical cells and HPV particles into the water. The sample tubes were sealed and the outsides of the tubes decontaminated with RNase A WA Y (Molecular Bioproducts, Inc) Hi) Preparation of HP V particles from Thin-Prep samples If Thin-Prep samples are used, cells taken from the cervix are rinsed in a solution of cellular preservative. Obscuring blood and mucus are machine-separated allowing the segregation and examination of the relevant cervical cells (Chacho et al. Cancer (2003) 99: 135-140; Weintraub A and Morabia (2000) J Diagn Cytopathol 22: 52-59).

Samples in distilled water were pipetted into a deep well microtitre plate and DNA was extracted according to a standard gene urine-swab DNA extraction protocol, for example using the protocol as follows.

DNA extraction was carried out using an automated CAS 1820 DNA extractor (X- tractor Gene, Corbett Life Sciences). Three hundred μl of the cell and HPV sample in distilled water for each specimen were loaded into wells of a lysis plate. Each sample was subjected to a standard DNA extraction protocol, which included 10 minutes incubation with 570μl of Lysis Buffer (Sigma), loading 570 μl of Lysis buffer treated mixture into a Whatman filter plate, the addition of 380 μl of propanol wash buffer (Sigma) for 15 seconds and removal of the solution by vacuum, the addition of 700 μl of 100% ethanol for 15 seconds and removal of the solution by vacuum, the addition of 100 μl of deionized water (Baxter) and incubation for 2 minutes followed by removal by vacuum, and elution of the DNA, which was then ready for amplification and detection. Example 2. Screening for the presence of HPV DNA in a sample HPV DNA present in samples was amplified and detected using an automated real time PCR system.

The oligonucleotide primers which were used were modified from the SPF primers described in Kieter et al. (supra), the entire contents of which is incorporated herein by cross reference. a) modified SPF Forward primer (5' to 3')

GCICARGGICAYAAYAATGG (SEQ ID NO: 1 ) b) modified SPR Reverse primer (5' to 3') GTIGTATCIACWACAGTAACAAA (SEQ ID NO:2)

These consensus sequences differed from those described by Kleter et al. in that instead of using several primer pairs the present technique requires only one pair. This is advantageous as it uses less reagents, and provides a decreased chance of misidentification.

Real time PCR was carried out using a Rotor-Gene ™ 3000 automated DNA detection system (Corbett Life Science, Inc). To set up the amplification reaction for the detection of Human Papilloma Virus, reagents were used in the volumes outlined in Table 1. Table 1

Reagents Final Reaction concentration volume (μl)

BioXact Master Mix Ix 10.0

Containing 4mM MgCl₂

SPFI Forward 0.5 μM 1

SPR Reverse 0.5μM 1

Syto 9 1.5 μM 1

MgCl₂ 1.5 μM 0.6

DNA template 5

Molecular grade H₂O 1.4

TOTAL 20

The thermal cycling protocol for the detection of Human Papilloma Virus using these primers was as follows: Denaturation step: 95°C (5 min) 1 cycle;

Cycling step 1: 95°C (15 sec), 48°C (15sec), 72°C (45sec) 20 cycles;

Cycling step 2: 95°C (15sec), 45°C (15sec), 72°C (45sec) 25 cycles.

Signal intensity threshold was determined manually.

As a rule of thumb, any specimen which showed an increase in the fluorescent signal in the green channel of 10% or more between cycles 25 to 41 was positive for HPV.

DNA extracted from Factor V Leiden samples were used as controls in the screening PCR reactions. A total of 320 specimens were screened using this protocol. Twenty specimens were from tampons, and 300 specimens were from Thin Prep™ specimens. All but four specimens were also tested using the Hybrid Capture 2 high sensitivity protocol.

Of the 320 specimens screened, the protocol described above identified 214 HPV- positive specimens, which equates to two-thirds of the screened specimens. In contrast, of the 300 specimens examined using the Hybrid Capture 2 technique, only 124 HPV positive specimens were detected.

Table 2: Percentage of HPV positives between Hybrid Capture 2 and modified

SPF primers

Hybrid Capture 2 % Mod. SPF Protocol %

Positive 134 42.41 214 66.88

Negative 182 57.59 106 33.13

TOTAL 316 320

This technique alone differs from other techniques which use a set of consensus or degenerate primers to amplify multiple HPV types as this technique recognises the presence of both low and high risk HPV types. This screening technique is also more sensitive than the use of GP5/GP6 and MYOl 1 primers.

Example 3. Identifying specific HPV types in a sample

A test to identify oncogenic HPV types was carried out for each of the original biological samples which was positive for HPV from the method of Example 2. HPV DNA present in the biological samples was amplified and detected by PCR using a Rotor-Gene™ 3000 automated DNA detection system (Corbett Life Science, Inc).

In the present example, a series of multiplex PCR reactions, using up to 4 primer sets and a single probe, was used to genotype the HPV DNA present in the sample. Each positive sample was thus effectively tested for each of the 17 high oncogenic risk HPV types examined, using at most a cocktail of 5 primer sets in any one test, so that competitive amplification of a particular HPV type DNA was minimised. The specificity of the reaction was provided by the primers, since each set of primers recognises only a single oncogenic HPV type. Once a specific primer-probe set cocktail produced a positive reaction, the test was repeated once more using single primer sets from that cocktail to type the HPV being tested.

The following HPV type-specific primers were used (described in the 5' to 3' orientation):

SDSHPV 16F:

GGA AAT CCA GTG TAT GAG CTT AAT GA (SEQ ID NO:3)

No. of Bases: 26

SDSHIPV 16R: CTC GTC CTC GTG CAA ACT TAA TC (SEQ ID NO:4)

No. of Bases: 23

SDSHPV 18F:

TGC AGAAAA CCA CAG ACA TAA GC (SEQ IDNO:5)

No. of Bases: 23 SDSHPV 18R:

TTT ATC CTA CAA TCC TCC ATT TTG C (SEQ ID NO:6)

No. of Bases: 25

SDSHPV31F:

AGC AGA CTG GTG GTT TTT ACA TTT C (SEQ ID NO:7) No. of Bases: 25

SDSHPV31R:

CCT CTT CCT CGT GCA AAT TTA ATC (SEQ ID NO:8)

No. of Bases: 24

SDSHPV33F: CAG GTAATT GTTTGT CCT ACG TCTATATCT (SEQ IDNO:9)

No. ofBases: 30

SDSHPV33R:

TCG GTTATC GTT GTCTGT CTGTAT G (SEQ IDNO:10) No. of Bases: 25

SDSHPV35F:

ACC CAG AGT ATG CGC TTA ATG ATA A (SEQ ID NO: 11 )

No. of Bases: 25 SDSHPV35R:

CCT CTT CCT CGT GCA AAT TTA ATC (SEQ ID NO: 12)

No. of Bases: 24

SDSHPV39F:

GAT TAT GTT ACA CGC ACA GGC ATA T (SEQ ID NO: 13) No. of Bases: 25

SDSHPV39R:

CAA TGT CAC GCG AAA TAC CCT AT (SEQ ID NO: 14)

No. of Bases: 23

SDSHPV45F: GCG CGC CAG TAG GTT AAA CT (SEQ ID NO: 15)

No. of Bases: 20

SDSHPV45R:

TGC ATA CTA CAC AGC ATC CAT TTT AC (SEQ ID NO: 16)

No. of Bases: 26 SDSHPV51F:

GGG CGG ATG TAT GCA CTA TGT (SEQ ID NO: 17)

No. of Bases: 21

SDSHPV51R:

TGT TAC AGC TGC AGC CAT TTT G (SEQ ID NO: 18) No. of Bases: 22

SDSHPV52F:

TTG TTT GTC CTG CAT CTG TAT CTA GTA A (SEQ ID NO: 19)

No. of Bases: 28

SDSHPV52R: TGG AGG TTT CGG TGC ATA GG (SEQ ID NO:20)

No. of Bases: 20

SDSHPV56F:

TTG TTT GCT TGT GTG TCA TGT TG (SEQ ID NO:21) No. of Bases: 23

SDSHPV56R:

AAA GGA TGT GGC TAT AAC AAA CCA A (SEQ ID NO:22)

No. of Bases: 25 SDSHPV58F:

GTG GGT AGT CGG GTA ATT GTA TGT C (SEQ ID NO:23)

No. of Bases: 25

SDSHPV58R:

TGG CCT CGG TGG TCT TTG (SEQ ID NO:24) No. of Bases: 18

SDSHPV59F:

GGT GGT AAT GGT AGA CAG GAT GTT C (SEQ ID NO:25)

No. of Bases: 25

SDSHPV59R: GTT ATC TGG AAG GCC AAA TTT ATT G (SEQ ID NO:26)

No. of Bases: 25

SDSHPV66F:

TTT GTC TGT GTG TGT GCC ATT TT (SEQ ID NO:27)

No. of Bases: 23 SDSHPV66R:

ATG ATG TAG CCA CAA CAA ACC AA (SEQ 1 D NO:28)

No. of Bases: 23

SDSHPV68F:

GCC GCA AGC AGG ACA TTC (SEQ ID NO: 29) No. of Bases: 18

SDSHPV68R:

TTT ATT AGG ATC AGG TAG GGA AAT CC (SEQ ID NO:30)

No. of Bases: 26

SDSHPV70F: GCC GCA AGC AGG AAA TAC C (SEQ ID NO: 31 )

No. of Bases: 19

SDSHPV70R:

CGG AAG GCC AAA CTT ATT AGG AT (SEQ ID NO:32) No. of Bases: 23

SDSHPV73F:

CTC TGT CTC GTA AGT TTC CAC TTC AG (SEQ ID NO:33)

No. of bases 26 SDSHPV73R:

AGA AGT ACA TCT AAA CGA AGA CTG TTT GA (SEQ ID NO:34)

No. of bases 29

SDSHPV82F:

CCT TCC ACA GTC TCC ACT TGT G (SEQ ID NO:35) No. of bases 22

SDSHPV82R:

CGG TAA AAG GAG ATT ACT GGA CAG TT (SEQ ID NO:36)

No. of bases 26 where for each of the primers identified above as "SDSHPVXXY": HPVXX = HPV type and

Y = F for forward primer or R for reverse primer

The following HPV probes were used:

HPV33/52/58 Probe: SEQ ID NO:37

CCA CTA CTG AAA CTG CTG HPV 18/45/51 Probe: SEQ ID NO:38

CCG AAA TCG GTT GCA C

HPV39/59/68/70 Probe: SEQ ID NO:39

AAG GTG TCT GCA TAT CA

HPV16/31/35 Probe: SEQ ID NO:40 CTG GAA ATC CTT TTT CTC

HPV56/66 Probe: SEQ ID NO:41

CCC GCT TTT GCT ATC T

HPV 73/82 Probe: SEQ ID NO:42

ATT GCC ATA TCC ACT GTC Each probe comprised a Minor Groove Binding, Non-Fluorescent Quencher moiety

(Applied Biosystems, Inc.) at the 3' end, and a unique fluorescent reporter dye, such as 6 carboxyfluorescein (6FAM, Applied Biosystems, Inc.), VIC or NED (Applied

Biosystems, Inc), at the 5' end so that binding of each different probe in a cocktail could be detected in a different colour channel. While up to five probes were used simultaneously, more commonly only two or three probes were used in a cocktail.

To set up the amplification reaction for the typing of HPV, the following reagents were mixed together in the volumes outlined in Table 3. Table 3

The thermal cycling protocol for the detection of Human Papilloma Virus using these primers was as follows:

Denaturation step: 95⁰C (10 min) 1 x cycle

Cycling step: 95°C (15 sec), 60°C (45 sec) 40 x cycles.

As a rule of thumb, any specimen which showed an increase in the fluorescent signal in the respective channel of 10% or more between cycles 20-41 was considered positive for a specific HPV type. A signal intensity threshold was determined manually.

Fourteen high risk HPV types were examined in this assay. Of the total 214 specimens that tested positive with the SPF protocol, 103 specimens were tested positive to one of the high risk HPV types examined, the remaining 11 types were low-risk, which is in accord with the general incidence of high and low risk types in the population tested.

Of the high-risk HPV types which were tested for, types 16 and 52 were the most common, followed by 51, 18, and 58 (Table 4). Table 4:

HPV type 16 was found in 28 of the 103 specimens which were typed for HPV, accounting for 27.18% of positive, typed specimens. Similarly, HPV type 52 was found in 24 specimens (23.33% of positive, typed specimens).

Of the 103 specimens which were typed, 7 tested negative for high-risk HPV types by Hybrid Capture 2 These false negatives from the Hybrid Capture-2 technique included types 16, 18, 45, 51, 52, 58, and 68. One of these specimens had multiple HPV infection of types 16, 45, and 52.

The typing of specimens showed that co-infection with multiple HPV types was common. Thirty of the 103 positive, typed specimens tested positive for more than 1 high- risk HPV type. Of these 30 specimens, HPV type 16 was clearly the most common, being detected in 16 of the 30. Type 52 was detected 10 times, and types 18 and 51 were both being detected in 8 specimens (Table 5).

An analysis of HPV types most commonly found in those individuals infected with a single type reveals that HPV type 52 was the most prevalent (13.59%), followed by HPV type 16 (11.65%). Furthermore, in specimens with multiple infections it was most common for a specimen to have 2 types (19/30). Table 5: Incidence of multiple HPV types

Of the 214 positive specimens identified in Example 2, 111 specimens could not be typed by the fourteen type-specific primers examined in Example 3. Table 6 shows the number of specimens that tested negative for Hybrid Capture 2, but positive by the SPF protocol that could not be typed, compared with the specimens that tested positive for both that could not be typed either. Table 6

%

Specimens -ve by Hybrid Capture 2, +ve by SPF that were not typed 72 33 .64

Specimens +ve by Hybrid Capture 2, +ve by SPF that were not typed 39 18 .22

11 1

Example 4. Use of Tampons for sample collection To increase the ease of use of this assay in a clinical setting, the method was further extended to include DNA extracted from tampons. Twenty samples were collected and of these, 15 tested positive by the SPF protocol described in Example 2. Sixteen of these specimens had been tested by the Hybrid Capture-2 technique.

Of the tested samples, the modified SPF protocol described in Example 2 detected 5 more positive specimens than the Hybrid Capture-2 technique. The HPV type-specific primers described in Example 3 typed nine of the twenty tampon collected specimens, 7 of which also tested positive by the Hybrid Capture-2 technique. The five samples which were not typed as high risk variants appear to be low oncogenic-risk HPV types. Table 6

Six of the twenty tampon provided specimens could not be typed by the 14 type- specific primers described in Example 3. Of these 6, 3 specimens tested negative and 3 specimens tested positive using the Hybrid Capture 2 technique.

The HPV type-specific primers described in Example 3 typed nine of the twenty tampon provided specimens; 7 of these specimens also tested positive by Hybrid Capture 2 technique and the other 2 were not tested by Hybrid Capture 2.

Although the invention has been described with reference to certain embodiments and examples it will be understood that variations in keeping with the disclosure and spirit of the invention are also contemplated and encompassed herewith.

Claims

What is claimed is:

1. A method of identifying and distinguishing between human papilloma virus types in a biological sample, comprising the amplification and detection of human papilloma virus DNA in the biological sample using PCR with human papilloma virus type-specific primers.

2. The method according to claim 1, comprising the additional step of screening for the presence of human papilloma virus in the biological sample.

3. A method according to claim 1, wherein the method is capable of identifying and distinguishing between multiple human papilloma virus types in a sample infected with more than one human papilloma virus type.

4. A method according to claim 1, wherein the method is capable of identifying and distinguishing between multiple high oncogenic risk human papilloma virus types in a sample infected with more than one human papilloma virus type.

5. A method according to claim 1, wherein the human papilloma virus type-specific primers include any one or more nucleotide sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ 1D NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

6. A method according to claim 1, wherein the amplification of human papilloma virus DNA is identified by one or more probe nucleotide sequences selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID N0:41, and SEQ ID NO:42.

7. A method according to claim 6, wherein the one or more probe nucleotide sequences comprise a minor groove-binding moiety.

8. A method according to claim 1, wherein the human papilloma virus types are high oncogenic risk human anogenital papilloma virus types.

9. A method according to claim 1, wherein the biological sample is a cervical sample.

10. A method according to claim 1, wherein the biological sample is obtained from a tampon.

11. A method according to claim 2, wherein the screening for the presence of human papilloma virus in the biological sample is by amplification and detection of human papilloma virus DNA.

12. A method according to claim 11, wherein the amplification and detection of human papilloma virus DNA is by real time PCR.

13. A method according to claim 12, wherein the amplification of human papilloma virus DNA is detected using Syto 9 dye.

14. A method according to claim 11, wherein the amplification of human papilloma virus DNA utilises at least one consensus primer for multiple human papilloma virus types.

15. A method according to claim 14, wherein the at least one consensus primer is selected from the nucleotide sequence SEQ ID NO:1 and/or SEQ ID NO:2.

16. A method according to claim 2, wherein the screening for the presence of human papilloma virus is carried out before the identifying and distinguishing between human papilloma virus types in the biological sample.

17. A kit for identifying and distinguishing between human papilloma virus types in a biological sample by PCR, comprising human papilloma virus type-specific primers.

18. A kit according to claim 17, wherein the human papilloma virus type-specific primers are selected from the group consisting of any one or more of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10. SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

19. A kit according to claim 17, comprising at least one consensus primer for is multiple human papilloma virus types.

20. A kit according to claim 19, comprising at least one oligonucleotide probe selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 , and SEQ ID NO:42.

21. A method of screening for the presence of human papilloma virus in a biological sample by amplification and detection of human papilloma virus DNA using PCR and at least one human papilloma virus consensus primer which is selected from the nucleotide sequence SEQ ID NO:1 and/or SEQ ID NO:2.

22. A method according to claim 21, comprising the step of identifying and distinguishing between human papilloma virus types in the biological sample by the amplification of human papilloma virus DNA using PCR with human papilloma virus type-specific primers.

23. A method of diagnosis of HPV infection in a subject, comprising obtaining a biological specimen which is suspected of containing HPV from a subject and amplifying and detecting human papilloma virus DNA using PCR with human papilloma virus type specific primers.

24. A method according to claim 23, wherein the diagnosis of HPV infection identifies a predisposition for HPV-associated neoplasia.

25. A method of identifying a persistent or recurring HPV infection, comprising comparing the HPV types present in two or more temporally separated biological samples from a subject wherein the HPV types are identified and distinguished by amplifying and detecting type-specific human papilloma virus DNA in the biological samples using PCR with human papilloma virus type-specific primers.

26. A method of screening for HPV types in a biological sample, comprising

(i) screening the biological sample for the presence of HPV using real time PCR and consensus primers specific to HPV; and (ii) where HPV was detected in (i) screening for HPV types using PCR and HPV type-specific primers

27. A consensus primer for screening for the presence of HPV in a biological sample, the consensus primer consisting of a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2.

28. An HPV type-specific primer for the identification of specific HPV types in a biological sample, the HPV type-specific primer consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ 1ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ]D NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ 1ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.